Synthesis of biotin



Patented Nov. 22, 1949 SYNTHESIS OF BIOTIN Moses Wolf Goldberg, Upper. Montclairyand Leo H. Sternbach, Montclair, N; J:, assignors to Hoifmann-La Roche Inc., Roche Park, Nutley,

N. J a corporation of New Jersey No Drawing. Application M 31 1946, Serial No. 673342 v l 8 Claims. lamen 309) Our invention relates to a new synthesis of d,l-biotin. The synthesis is characterized by the use of a meso-diaminosuccinic acid derivative as a starting material. This is converted into the corresponding imidazolidone- (2) -cis-4,5-dicarboxylic acid, then the sulfur-containing ring is formed, and finally the typical biotin side chain introduced. Our synthesis differs thus in several respects from the two already published syntheses of biotin by Harris et al., J. Am. Chem. Soc., 66, 1756, 1757 (1945); 67, 2096 (1946), and by A. Griissner, O. Schnider and J. P. Bourquin, Helv. Chim. Acta, 28, 517 (1945) An important point is, that our easily accessible starting material 15 The flow sheet shows the steps of the already contains the two amino groups in the synthesis. k 1

R=benzyl CO COOH Br I Br l ]lHR I R 3 NR QH=CH H- oH CH-CH B t .fcoon .OOH (LJOOH $0011 )0011 (50011" $0011 I 11 III I 1 Iv i g v t 1 .1 -f RN/ NR RN it co (tn- 511 I oo 6 1-411: RN/ \IIIR Moon (to XH'RN/ R (oH,)iocHl H-CH 0 VI .H-CH I s 0H v (to (to i I 4 :11. 30 VIII ii I iT 0 s v v v. V VII 7 g RN NR RN NR one-o til-coon j Es CH2 t'H-omomnocm VIa VIIIa co 5 co oo R \NR 7 RN NR HN \NR (|3H+H tH -c11 l me-on cm o=c cHl ,ocH3 I Hi 2)i0GHi H| (En 0119 00113 v p p I s s s IX 7 x XI C0 fisf3 HN/ \NR E P. HN NR tn, oH(oH, .-Br OH; GH-(CHzM-CN H, oaqonm-ooon S S S X11. 7 I a XIII 1 XIV f3 HN NH 5/ n. on, IJH-(CHzh-COOH same spatial arrangement as the two amino groups present (in substituted form) in the biotin molecule, the meso-conflguration in diaminosuccinic. acidderivatives corresponding to known bis benxylaminossuccinic acid (III). This a cis-structure of the two amino groups in a ring compound asin biotin). This approach to the synthesis of biotin is basically new. It is in contrast to the known syntheses, which introduce one orboth amino groups at a later stage of the synthesis intoa preformed sulfur-containing ring an approach-Which permitsformation of the corresponding cisand trans-isomers. 'rnessj must be separated thus complicating the syn thesis and affecting the overall yield of biotin. Ourprocess avoids the formation of the bio-} logically inactive trans-isomers of biotin (allo-f biotin and epi-allobiotin), and gives as the final product, pure d,l-biotin.

Fumaric acid (I) is brominated in the usual way, to yield meso-dibromosuccinic acid (II), which in turn can be easily converted into the acid is reacted in alkaline solution with phosgene, to yield the 1,3-dibenzyl-imidazolidone-(2)-cis- 4,5-dicarboxylic acid (IV). This compound and all the following ones except d,l-biotin are novel. The 1,3-dibenzyl-imidazolidone- (2) -cis-4,5-dicarboxylic acid (IV) is heated with acetic anhydride, and the anhydride (V) formedis reduced with zinc, acetic acid and acetic anhydride to yield in an unusual reaction the acetate of the cyclic form of the corresponding aldehydo acid, the 3,4(1',3-dibenzyl 2-keto-imidazo1ido) -2- keto--acetoxy-tetrahydro-furane (VI).

Compound (VI), or the corresponding free aldehydo acid (V'Ia), the 1,3-dibenzyl-cis-4-carboxy-5-formyl-imidazolidone-2, which can be obtained from the'acetate (-VI) by saponification, were found to be suitable materials for the introduction of the sulfur atom into the molecule. The acetate, or the free aldehydo acid, is treated with hydrogen chloride and hydrogen sulfide in an organic solvent; the mixture is concentrated in vacuo, and reacted with an alcoholic solution of potassium hydrosulfide. The reaction mixture is acidified in the cold, extracted with ether, the ether extract concentrated in vacuo, and the residue reduced with zinc and acetic acid. The reduced reaction mixture is filtered, the filtrate concentrated in vacuo, and the residue extracted with ether. The ether solution is then concentrated, and after -60 hours the thiolactone (VII) crystallizes out.

iis thiolactone (VII) reacts very well with methoxybutyl-magnesium bromide. The product obtained corresponds to formulae (VIII) or (VIIIa). It can react in both tautomeric forms. Upon heating with acetic acid this product (VIII or VHIa) loses water and is converted into an unsaturated compound of the formula (IX).

This compound is hydrogenated with a palladium catalyst at room temperature and atmospheric pressure. The hydrogenation product (X) is then reacted in liquid ammonia with 2 atoms of sodium which causes the loss of one of the benzyl groups and the formation of a high melting well crystallizing N-monobenzyl derivative (XI). Whereas formula (XI), et seq., illustrate compounds wherein the benzyl is shown in the 3' position,v it is to be understood that the benzyl group can appear in one of the positions 1 and 3'.

Compound XI is treated with hydrogen bromide in acetic acid to yield a bromo derivative (XII), which is converted into the nitrile XIII, and finally into monobenzylbiotin (XIV). This compound is reacted with sodium in liquid ammonia, and loses its benzyl group to yield d,1-biotin The monobenzylbiotin can also be debenzylated by catalytic hydrogenation with a palladium catalyst in presence of mineral acid,

The crystalline d,l-biotin (XV) has a melting point of 232-234. The activity of our d,l-biotin in the Saccharomyces cerem'siae test was of di-biotin; with Lactobacillus casei an activity of 51% was found. The methylester had a melting point of 134436".

EXPERIMENTAL PART (All melting points are corrected) Preparation of bis-benzylamino-succinic acid (III) To a warm solution of 300 grams meso-dibromosuccinic acid (prepared according to Org. Synththe anhydride.

Coll., vol. II, page 177) in 2 liters of absolute alcohol are added 900 cc. of benzylamine. The mixture is stirred and refluxed for 5-6 hours. 300 cc. of concentrated hydrochloric acid, 50 cc. of concentrated acetic acid, and 500 cc. of water are then added, and the slightl acidic mixture (pH 5.0) is concentrated, by distilling off 500-1000 cc. of alcohol. The reaction product is thus obtained in the form of an easily filterable precipitate. The mixture is kept overnight in the refrigerator, then the precipitate is filtered off and washed several times with water. If the precipitate is not white and does not filter well, it is dissolved in a hot solution of 114 grams potassium hydroxide in 1.5 liters of water. This solution is then decolorized with charcoal, filtered, boiled up, and treated with 170 cc. of concentrated acetic acid. The mixture is left for 1 hour on the steam-bath, and then kept overnight in the refrigerator. The reaction product, which is thus obtained in form of white crystals, is filtered off, washed with water and alcohol, and then dried. Melting point 224-225".

1 ,3-dibenzyZ-imidazolidone- (2 )-cis-4,5-dicarborylic acid (IV) and its anhydride (V) To a stirred, ice cooled solution of 6418 grams 1.98 moles) of bis-benzylaminosuccinic acid in 2 liters 3 N potassium hydroxide, are added in small portions (within 1 /2 hours) 1.6 liters of a 3.75 molar solution of phosgene in xylene (:6 moles C0012) and 2.7 liters 6 N potassium hydroxide (:14 moles). The mixture is then acidified with concentrated hydrochloric acid, and the formed precipitate filtered 01f and Washed with water.

The precipitate is then washed thoroughly with hot alcohol, and the aqueous filtrate is extracted several times with ethyl acetate. The alcoholic solution and the ethyl acetate extract contain all the imidazolidone-dicar'boxylic acid formed. The alcohol insoluble part of the precipitate is pure starting material (220 grams=0.65 mole). The alcoholic and ethyl acetate solutions are taken to dryness, and the residue is refluxed with benzene until it becomes completely crystalline. The cooled mixture is filtered; the main part of the dibenzyl-imidazolidone-dicarboxylic acid (IV) remains on the funnel (283 grams=.08 mole). It can be recrystallized from ethyl acetate. Prisms, melting first at 167, then resolidifying again and melting at 236.

The benzene mother liquor is concentrated in vacuo, and the rest of the reaction product is isolated in form of its anhydride in the following Way: The oily residue is refluxed with acetic anhydride, then the mixture is concentrated and benzene is added. The anhydride (V) formed crystallizes in nice needles. Melting point 236- 237.

Acetate of the cyclic form of 1,3-dibeneyZ-cz's-4- carbozt'y-S-fOTmyZ-imidazOZidOne-Z; 3,4-(1,3'- dibeneyZ-2'-keto imidazolido) 2-keto-5-acetory-tetrahydrofurane (VI) A mixture of grams of the anhydride (V) 0f 1,3-dibenzyl-imidazolidone-cis-.4,5-dicarboxylic acid, grams zinc dust, 100 grams zinc powder (40 mesh), 1500 cc. acetic anhydride and 500 cc. acetic acid is stirred and refluxed for 10-15 hours. The dicarboxylic acid (IV) can be used instead of In that case the acid is first mixed with the acetic anhydride, refluxed for 10 minutes and then the other constituents are added. The amounts of acetic acid and anhydride can be reduced to about /2 without affectingthe mea er- 51 yield. Iftoo'little is used the yield is reduced; After that time, the mixture is cooled, filtered and the precipitate on the funnel washed with ethyl acetate. The filtrate is concentrated in vacuo, and the oily or partly crystalline residue is treated with ice water and ethyl acetate, until all the organic substance is dissolved. The ethyl acetate layer is washed with Water, dried with sodium sulfate and concentrated in vacuo. The oily or partly crystalline residue is boiled up with acetic anhydride, in order to reconvert any of the compound decomposed during the treatment with water into the cyclic acetate, The mixture is again concentrated in vacuo. Xylene is then added to the residue, and part of it is distilled off in vacuo, then ether and petrol-ether are added. The reaction product separates in fine needles or prisms, which melt at 103-104. After drying the melting point is 124-125".

1,3-dibenzyl-cis-4-carboxy-5-formylimz'dazoZidone-Z (VI a) An excess of 3 N sodium hydroxide solution is added to a solution of the acetate of the cyclic form of 1,3-dibenzyl-cis-4-carboxy-5-formylimidazolidone-Z (VI) in dioxane. After 2 minutes the mixture is acidified with dilute sulfuric acid and extracted with ether. The ether extract iswashed, dried with sodium sulfate, and concentrated in vacuo. The residue is recrystallized from a mixture of acetone, ether and petrolether.

' The free aldehydo acid (VIa) crystallizes in colorless needles or prisms melting at 109-110". It forms a 2,4-dinitrophenylhydrazone, which crystallizes from a mixture of dioXane, benzene and petrol-ether in orange prisms melting at 2235-2459. r

The free aldehydo acid (VIa) can be reconverted into the acetate of the cyclic form (VI) by treating it with acetic anhydride or acetyl chloride.

Thiolactone of 1,3-dibenz'JZ-cis 4 embassy-- mercaptomethyl-imidaeolidone-2 3,4-(1,3-dibenzyl-Z'-ket0-imidazolido) 2-keto-thiophane (VII) A. A vigorous stream of hydrogen chloride and hydrogen sulfide is passed through a suspension of 150 grams of the acetate of the cyclic form of 1,3-dibenzyl-cis-4-carboxy 5 -iormyl-imidazolidone- 2 (VI) in 850 cc. absolute alcohol cooled to to After about 1% hours, most of the starting material is dissolved. To complete the reaction, hydrogen chloride and hydrogen sulfide are passed through the solution for another hour. The solution is then concentrated in vacuo, at the lowest possible temperature andwith exclusion of moisture. Some toluene is added to the residue and distilled off in vacuo, thus removing most of the hydrogen chloride still present. B. A vigorous stream of hydrogen chloride and hydrogen sulfide is passed through a solution of 150 grams 1,3-dibenzyl-cis-4-carboxy-5-formylim'idazolidone-2 (VIa) in 850 cc. absolute alcohol cooled to -10 to -20. After 2 hours the solution is concentrated in vacuo at the lowest possible temperature and with exclusion of moisture. Some toluene is added to the residue and distilled off in vacuo, thus removing most of the hydrogen chloride still present.

C. The residual oil from reaction A or reaction B is dissolved in 1200 cc. of a suspension of potassium hydrosulfide in alcohol. (This suspension is prepared by saturating a mixture of 672 6 grams potassium hydroxide and 4 liters of alcoho with hydrogen sulfide.) The mixture is left at room temperature for 15 hours,then refluxed for 1 hour, poured on ice and acidified with dilute sulfuric acid. 7 The organic part is extracted with ether, the solution is dried with sodium sulfate and concentrated in vacuo. The residue is reduced in a stirred refluxing mixture of 900 grams zinc dust, grams granulated zinc (20 mesh) and 2.3liters acetic acid. After 4hours,the solution is filtered, and the mixture'of zinc and zinc acetate remaining on the funnel is washed with ether and water. i The filtrate is concentrated in vacuo and diluted with ether and 40% sulfuric acid until clear separation takes place. The ether layer is separated, washed with water and concentrated in vacuo to a small volume. After 24-48 hours the mixture becomes crystalline. It is then diluted with some more ether and filtered. Melting point 123-124".

The thiolactone can be recrystallized from a mixture of acetone, ether and petrol-ether. The pure compound forms colorless plates melting at 126-127". It is soluble in strong alkali, and gives a positive mercaptan test with sodium nitroprusside.

3,4-(1,3-dibeneyl-2-keto-imidazolido) 2 hydromy 2 (o-methowybutyl) -thiophane (VIII) 01" 1,3 dibenzyZ-cis 4 mercaptomethyl-5-(omethomy-oaleryl)-imidazolidone-2 (VIIIa) A Grignard solution prepared from 8.3 grams (50 m. moles=6.5 cc.) 4-methoxybutylbromide and an excess of magnesium (2.4 grams) in 15 cc. ether and 5 cc. benzene is diluted with benzene, poured off the unreacted magnesium and added dropwise (in about 30 minutes) to a boiling, stirred solution of 13.5 grams (40 m. moles) of the thiolactone (VII) in cc. benzene. The solution is then kept refluxing for another 2 hours. The mixture is decomposed with ice and dilute sulfuric acid, the organic layer is separated and concentrated in vacuo. The residue is dissolved in methanol and heated to 50 C. with an excess of aqueous sodium carbonate solution. Ether and water are added, and the two layers separated. The alkaline aqueous solution contains the unreacted thiolactone (VII), which is extracted and recovered after acidification (1.0 gram). The ether layer, containing the reaction product, is dried, and concentrated in vacuo. The residue is recrystallized from ether, petrolether.

The product is soluble in strong alkali and gives a positive m-ercaptan test with sodium nitroprusside. It canbe recrystallized from a mixture of acetone, ether and petrol-ether. Prisms. Melting point 110.5-112 C.

. 3,4- (1',3'-dibenzyl-2-7cet-O-imida20lido) -2(wmcthoxy-butylidene) -thiophane (IX) A solution of 20.0 grams of the above described oompoundWIIIe' VIIIa) in 100 cc. acetic acid is is refluxed for 1 /2 hours. The solution is concentrated in vacuo, the residue is dissolved in ether and the solution is washed with dilute sodium carbonate solution. The ether solution is dried and concentrated in vacuo. The oily residue represents the unsaturated w-methoxy-butylidene compound (IX).

3,4- (1 ',3'-dibeneyZ-2'-keto-imidaeolidol -2 (wmethoxybutyl) -thiophane (X) The oily w-methoxy-buty1idenecompound (IX),

obtained in the previous experiment, is dissolved 7 111 150 cc. methanol and hydrogenated in presence of 4 grams prehydrogenated palladium oxide at room temperature and atmospheric pressure. The calculated amount of hydrogen is taken up in 30 hours, the catalyst is filtered off and the solution is concentrated in vacuo. The time required for the hydrogenation depends on the purity of the product and varies therefore. Other palladium catalysts, for example Pd-charcoal and Pd-barium sulfate, can also be used. The hydrogenation product solidifies after a few days. It has a low melting point and is difiicult to recrystallize. The crude compound is therefore used for the next step. Pure crystalline preparations can be obtained by slow recrystallization from petrol-ether.

3,4- (N -monobeneyl-2-7ceto-imidazolido) -2 (wmethoxybutyl) -thiophane (XI) A solution containing 6.84 grams (16.8. moles) of the crude w-methoxybutyl compound (X) in 20-30 cc. Xylene is added to 20-30 cc. of liquid ammonia cooled in a dry ice bath. To the stirred mixture is added sodium (in form of small pieces) until the blue color persists (about 34 m. moles=0.78 gram sodium are used). The last excess of sodium is destroyed with ammonium chloride, the ammonia is evaporated, and dilute sulfuric acid and ether are added. The mixture is stirred for a while and then filtered. The bulk of the reaction product remains on the funnel; a smaller part is dissolved in the ether layer of the filtrate. The latter is separated (if some product precipitates ethylacetate is added), dried and concentrated in vacuo. The residue is recrystallized from acetone-ether, together with the product obtained by filtration.

The product can be recrystallized from a mixture of acetone, ether and petrol-ether, yielding fine needles melting at 143-144.

3,4- (N -benzyZ-2'-7ceto-imida2olido) -2- (w-bromo-.

butyl) -thiophane (XII) A solution of 4.6 grams 3,4-(N-benzyl-2-ketoimidazolido) -2- (w-methoxybutyl) -thiophane in 40 cc. of 48% hydrogen bromide in acetic acid is heated in a sealed tube to 90 (bath temperature) for 45 minutes. The purple solution is concentrated in vacuo, the residue dissolved in ethyl acetate, and treated with water. Ether is added to the mixture, and the precipitated crystalline bromo-compound filtered off, and washed with water and ether.

The filtrate is diluted, if necessary, with some ethylacetate to dissolve some newly precipitated material. The organic layer is then separated and concentrated in vacuo. The crude material on the funnel and the residue from the organic layer are combined and used for the next step. The bromo-compound can be purified by dissolving the crude material in acetone or methanol. Ether and petrol-ether are added to such a solution to precipitate some dark impurities. The filtered solution is then concentrated, until crystallization begins. After some standing, other is added, and the precipitated needles are filtered off. Melting point 168-169.

3,4- (N -ben.2yl-2 -keto-imzdazlido) -2- (ti-cyanobutyl) -thiophane (XIII) A solution of 5 grams potassium cyanide in cc. water is added to a solution of 4.5 grams of the crude bromo-derivative (XII) in 200 cc. methanol. The mixture is refluxed for hours, concentrated in vacuo, and the residue treated with water and ether. The precipitated nitrile is filtered" oiI, and the aqueous solution extracted with ethyl acetate. The organic layer is con-' centrated in vacuo, the residue combined with the precipitated material, and used together for the next reaction. The nitrile can be recrystallized from a mixture of methanol, ether and petrol-ether, yielding needles, melting at 169.5 171".

3,4 (N benzyl 2' keto imidaaolido) 2 (wcarborybutyl) thiophane; N-benzyZ-dJ-biotin (XIV) The crude nitrile (XIII) from the preceding experiment is dissolved in a mixture of 300 cc. methanol and cc. 3 N aqueous potassium hy-. droxide, and heated on the steam-bath for 24 hours. The mixture is concentrated in vacuo, and the residue treated with water and ether. The precipitate is filtered oil, and the alkaline solution extracted in the cold with some ethyl acetate. Some unchanged nitrile can be recovered from the precipitate and the ethyl acetate extract. The alkaline aqueous solution is then acidified, and the precipitated Nbenzyl-d,l-biotin filtered ofi. The mother liquors are extracted with ethyl acetate, to yield another smaller portion of the reaction product. The product crystallizes very Well from methanol, if necessary with the addition of some ether. Thin plates, melting at 175-176.

The methyl ester Was prepared by reacting the free acid in methanol with a diazo-methane solution in ether. The ester can be recrystallized from a mixture of methanol, ether and petrolether. Fine needles melting at 169.5-1705.

d,l-Biotin 1.45 grams (=4.27 m. moles) pulverized monobenzyl-biotin are suspended in about 100 cc. liquid ammonia. Sodium is added in small pieces, until a persistent blue color appears (about 13-17 m. moles=0.3-0.4 gram of sodium are needed). The ammonia is evaporated, and ice, ether and dilute hydrochloric acid are added to the, residue. The acid solution is, filtered, and the residue on the funnel dissolved in a large amount of hot Water and hot ethyl acetate or chloroform. These solutions are mixed with the first filtrate, and the two layers are separated. The ethyl acetate or chloroform layer contains the unchanged starting material. The aqueous part contains all the d,l-biotin formed and inorganic salts. It is concentrated in vacuo, and the precipitated d,l-biotin is filtered off. The biotin is obtained in form of fine needles melting at 228-230". The sample for analysis was purified by dissolving in 20 parts hot 80% acetic acid and diluting the solution with the equal amount of Water. Fine needles melting at 232-234.

Anal. Calcd. for C10H16O3N2SZ C, 49.16; H, 6.60%. Found: C, 49.33; H, 6.49%.

It will be understood that in carrying out our new biotin synthesis, as disclosed herein, the

nature of the intermediates can be varied within.

the scope of the known art. Thus, instead of the acetate of the cyclic form of 1,3-dibenzyl-cis-4- carboxy-5-formyl-imidazolidone-2 (VI), the corresponding homologous lower aliphatic acyl-derivatives can also be used, c. g., the propionate or the butyrate. These acyl-derivatives can be prepared in the same Way as the acetate, viz., by carrying out the reduction of the anhydride (V) of the 1,3-dibenzyl-imidazolidone-(2) cis 4,5- dicarboxylic acid with zinc in the presence of the corresponding lower aliphatic acid and its anhydride. Furthermore, instead of using w-methoxybutylmagnesium bromide for the introduction of the butyl side chain into 3,4-(1',3'-dibenzy1- 2' keto imidazolido) 2 keto thiophane (thiolactone VII), organo-metal derivatives of other lower wall:oxy-buty1-halogenides can be employed as Well for the same purpose.

It will also be understood that the specification and claims embrace all tautomeric forms of the compounds named or shown. Moreover, it will be understood that the monobenzyl compounds, described herein, contain the benzyl group either in the 1' or 3 position, the formulae showing the benzyl in a fixed position merely for the sake of convenience.

We claim: 1. Compounds of the general formula AN/ \NB tH tH Hz 1 wherein A and B are members of the group consisting of hydrogen and benzyl, there being at least one benzyl substituent, and X is a member of the group consisting of :CO =C(OH) -a1koxyalkyl =C=CH-alkoxyalkyl =CH-alkoxya1kyl =CH-halogenoalkyl =CI-l-cyanoalkyl =CI-I-carboxyalky1 2. 3,4 (N-benzyl 2' keto imidazolido) 2- (w-bromobutyl) -thiophane.

3. 3,4 (N benzyl 2' keto imidazolido)- 2- (w-cyanobutyl) -thiophane.

4. N-benzyl-dJ-biotin.

5. Process for producing d,1-biotin which comprises reacting meso-bis-benzy1amino-succinic acid with phosgene to form 1,3-dibenzyl-imidazolidone-(Z) -cis-4,5-dicarboxylic acid, converting said compound to its anhydride, reducing said anhydride in the presence of an anhydride of a lower aliphatic acid to yield a corresponding 3,4 (1,3 dibenzyl-2 keto-imidazolido) 2- keto-5-acyloxy-tetrahydrofurane, converting the latter compound to 3,4-(1,3'-dibenzyl-2'-ketoimidazolido)-2-keto-thiophane, treating the 3,4- (1,3 dibenzyl 2' keto-imidazolido)2 ketothiophane with an to alkoxybutyl magnesium halogenide to form a 3,4-(1,3-dibenzy1-2'-ketoimidazolido) 2 hydroxy-Z (w alkoxybutyl) thiophane, splitting out one molecule of water from said last mentioned compound to yield a 3,4 (1,3 dibenzyl 2keto-imidazolido) 2- (o-allzoxybutylidene) thiophane, hydrogenating said unsaturated compound to a 3,4=- l,3-dibenzyl 2' keto imidazolido) 2 (w-zalkoxybutyl) -thiophane, thereafter removing one benzyl group to yield a 3,4-(N-monobenzyl-Z-keto-imidazolido) 2 (w-alkoxybutyl) thiophane, and converting said latter compound into a corresponding compound of the group consisting of those having in place of the alkoxy radical a member selected from the group consisting of halogeno, cyano, and carboxy, and eliminating the benzyl group from the carboXy compound by reductive cleavage.

6. In a process of producing d,l-biotin, the steps which comprise converting a compound selected from the group consisting of 3,4-(1,3-di benzyl 2' keto imidazolido) 2 keto 5- acyloxy-tetrahydrofuranes, the acyl being lower aliphatic, and the corresponding free aldehyde 1,3 dibenzyl cis 4 carboxy 5 forrnyl-imidazolidone-Z, to 3,4-(l,3-dibenzyl-2-keto-imidazolido) -2-keto-thiophane, followed by treating the 3,4-( 1',3 dibenzy1-2 keto imidazolido)- Z-keto-thiophane with an w-alkoxybutyl-magnesium halogenide, to form a 3,4-(l',3-dibenzyl- 2 keto imidazolido) 2 (w alkoxybutyD- thiophane, splitting out one molecule of water from said compound to yield a 3,4-(l',S-dibenzyl 2' keto imidazolido) 2 (w-alkOXY- buty1idene)-thiophane, hydrogenating said unsaturated compound to a 3,4-(1,3-dibenzyl-2- keto imidazolido) 2 (w alkoxybutyl)-thiophane, thereafter removing one benzyl group to yield a 3,4-(N-monobenzyl-Z-keto-imidaaolido) z-(w-alkoxybutyl) -thiophane, and converting said latter compound into a corresponding compound of the group consisting of those having in place of the alkoxy radical a member selected from the group consisting of halogeno, cyano, and carboxy.

7. Process which comprises eliminating the benzyl group from N-benzyl-dJ-biotin by reductive cleavage.

8. Process of claim 7 involving the use of sodium in liquid ammonia.

MOSES WOLF GOLDBERG. LEO H. STERNBACH.

No references cited.

Certificate of Correction Patent No. 2,489,232 November 22, 1949 MOSES WOLF GOLDBERG ET AL.

It is hereby certified that errors appear in the printed specification of the above numbered patentrequirlng correction as follows:

Column 2, Formula IV, for that portion reading same column, Formula VIIIa, for that portion reading HSCH C read HiS'C'H CH;

column 3, line 11, for 3,4(1 ,3 -dibenzyl read 3,4-(1,3-d1lbenzyl; line 65, for dibiotin" read d-bz'otin; column 6, lines 60 and 61, for -2(w-methoxyread -2-( wmetho:ry; line 64, after acid strike out is; column 10, line 31, for imidazolido)-2 read im'idazolido)-2-hydroa:y2; i

and that the said Letters Patent should be read with these corrections thereinthat the same may conform to the record of the case in the Patent Oflice.

Signed and sealed this 28th day of March, A. D. 1950.

THOMAS F. MURPHY,

Assistant Oommiau'oner of mum. 

